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TISSUE MODIFICATIONS IN MONKEYS AS RELATED TO ABSORPTION, DISTRIBUTION, AND EXCRETION OF POLYCHLORINATED BIPHENYLS J. R. ALLEN. I). II. NullIIACK. .,nd I. C. list: Department of P>tth<itn\;\ University of Wim'nnnn Mrdu-nt .V< /<*>/ uti Regional Primate Rr'tarth Center University of Wiu-onun Madison, Wiuon.un 5J 706 Adult rhesus monkeys were given a single dose of I.S or 3.0 g of a poly chlorinated biphenyl (PCB) (Aiochior 1248) per kg body weight end sacrificed four days later. In addition, one monkey from each group was placed in a meta bolism cage and the excreta collected during <t iwo-week period and analyzed for PCB content. Over 90 percent of the single P( I) dosage was absorbed from the gastrointestinal tract and deposited in various tissues of the body. Elimination of the PCBs. primarily through the biliary system, occurred at a slow rate. The monkeys did not become obviously ill following ingestion of the PfB*. but de veloped moderate hepatic enlargement due prinurily to an increase in lipid droplets and proliferation of the endoplasmic reticulum of the hepatic cells. Gastric hypertrophy and hyperplasia and focal ulceration of ihe stomach lining were prominent lesions in these animals. The polychlorinated biphenyls (PCBs) have been used extensively for various industrial purposes during the past 40 years. Because of their chemical inertness these compounds are highly resistant to degradation in the environment. Through industrial accidents and improper disposal and usage, they have become global environmental contaminants. Ingestion of minute amounts of the PCBs have produced widespread deleterious effects on the wildlife population throughout the world (Holmes et aL l%7; Jensen el oL 1969, Koeman ct aL 1967, 1969). Industrial accidents leading to ingestion by man have led to serious illness and long-tasting morbidity (Kuratsunc </ aL 1972). The presently reported study was conducted to determine acute toxic effects on nonhuman primates of PCBs (Aroclor 1248') and to evaluate absorption, tissue levels, and routes of excretion of these compounds following exposure to a single dosage. This investigation was supported in part by U.S. Public Health Service grants LS-00472 and RR-OOI67, and the University of Wisconsin Sea Grant. Primate Center Publication No. 13-029. I Monsanto Company, St. Louis, Missouri. Archives of tnvironmentat Contamination and Toxicology, Vo). 2. No. I, 1974 CI974 by Springer-Vertag New York Inc. 86 MQNS 085747 Tissue Modifications in Monkeys us Related to I'C.Tl 87 Materials and methods Seventeen male rhesus monkeys, ranging in age from (wo to 2.5 years and weighing between three and 3.5 kg, were placed in three groups. The two experimental group* con sisting of six animals per group were given 1,5 or 3.0 g of a PCB (Aroclor I 2481 per kg body weight by gastric intubation. The third group of five animals served as controls. AH of the experimental animals were deprived of food for one day prior to intubation. Following the administration of the PCBs the animals were allowed to cal ad libitum for three days, after which five animals in each experimental group received no food for one day and were subsequently sacrificed. Blood studies including evaluation of white blood cells (WBC), hemoglobin, hematocrit, differential white edi counts, serum glutamic oxalacetic transaminase (SCOT), serum glutamic pyruvic transaminase (SGPI) i Henman and Frankel 1957), total protein (Werchselbaum 1946). scrum protein electrophoresis (Beckman, 1965), and blood urea nitrogen (BUN) (Bohoun i t ul. 1968) were done both prior to intubation and before each animal was sacrificed. Prior to death the animals were anesthetized, the jugular vessels severed, and the animals allowed to exsanguinate. Tissues were obtained immediately for microscopic and biochemical studies. For histnlogrca) evaluations, small sections of the tissues were placed in ten percent neutral buffered formalin for 24 hr. They were subsequently dehydrated, embedded in paraffin, sectioned at five microns, and stained with hematoxylin and eosiri (Aimed Forces Institute of Pathology 1960). The hepatic tissues employed in the electron microscopic studies were cut into small cubes and fixed in osmium tetroxide and buffered with veronal acetate (Caulfield 1957) for 1.5 hr. They were subsequently dehydrated through a graded series of ethanol and embedded in an epoxy resin mixture(Mollenhaucr 1964). Sections of the tissues were cut on an ultramicrotome, placed on uncoaled copper grids, stained with uranyl acetate, and examined with an electron microscope. Additional portions of the liver were homogenized at 0C with two volume* of 0.25 M sucrose with 0.010 M MgClj and 0.015 M KCI (SKM). Levels of protein (Lowry et at. 1951) and DNA and RNA (Munro and Fleck 1966) were determined on portions of the homogenate. Microsomes were isolated by centrifugation at 100,000 G for 90 min of the poslmitochondrial supernatant (which was obtained centrifugation at 9,000 G for 20 min), washed by homogenization with sucrose and reccntrifuged, then resuspended in SKM equal to three volumes of liver and stored at -70'C. The activities of aromatic hydroxylase, nitroreductase, A^demethyluse, esterase, and giucose>6>phosphatasc and concentrations of protein were determined by methods previously reported (Norback and Allen 1972). At the beginning of the experiment one animal from each of the two treatment levels was placed in a metabolism cage and the urine and feces collected prior to intubation and over a two-week period thereafter. In addition to the urine and feces from these two animals, the brain, liver, and kidneys were also obtained from all animals and analyzed for content of PCBs. Liver and kidney samples were homogenized in hexane (500 mg: 20 ml); urine was extracted in hexane (10 ml: 15 ml); and feces were desiccated over CaClj for one week, pulverized in a mortar and pestle, and extracted in acetone (1 gm:25 mi). Brain tissue was extracted in acetone (500 tng:20 ml) (Jennings 1968) and / HONS 085748 88 J. R. Alien cl ui. aU tissue extracts were subsequently evaporated under dry nitrogen with anhydrous sodium sulfate in a 40C water bath. Clean up of the extracted material prior 10 analysis was carried out in a disposable pipe* microcolunm (Curlev cl al. 1971) containing silica gel 60 (0.05-0.2 mm) by elution with all bcn/enc-hcxanc mixture. Levels of PCBs were quantitated in a Hewlett-Packard Research (7000) Chromatograph employing a *3Ni electron capture detector. The Pyrex glass tulumn packed with Gas Chrom Q (80-100 mesh) coated with two percent SH-30 was operated at l70r'C with argonmethane (95%-5%) as carrier gas at a flow rate of 40 cc/min. PCB recovery studies were conducted on control samples of tissue and excreta. The PCBs were added directly to the urine, dried feces, and tissue homogenates and sutv sequently handled in a manner similar to that previously described. Results Following the administration of 1.5 or 3.0 g per kg of body weight of the PC U mixture, Arodor 1248, to the experimental monkeys, there were no gross manifestations or illness during the subsequent four days and the general appearance of the animals remained unchanged. Body weight of the animals immediately before receiving the PCBs (3.14 kg 0.40 for the higher dosage group; 3.03 kg 0.33 for the lower dosage group) did not differ significantly from the body weight four days after PCB administration (3.02 kg l 0.46 for the higher dosage group; 2.97 t 0.28 for (he lower dosage group). T he animals lliat were allowed to survive for 14 days became increasingly anorectic and lethargic and at 14 days weighed 2.7 kg (after receiving 1.5 g PCBs,'kg) and 1.8 kg (after receiving 3.0 g PCBs/kg). As shown in Table 1 the experimental animals had slight but significant decreases in hemoglobin and hematocrit levels. The moderate leukocytosis was attributed primarily to an increase in neutrophils. There was also a slight rise in the level of SCOT, while the levels of SGPT were moderately reduced. The total serum protein level was slightly decreased. Levels of BUN were nut modified appreciably. Gross lesions of the external surface of the body or of the internal organs were absent at sacrifice on the fourth day following administration of the PCBs. Microscopic ex amination of the tissues revealed a moderate hypertrophy and hyperplasia of the gastric mucosa and the presence of isolated mucinous cysts within the epithelium of ihe stomach. In addition, there was an occasional penetration of the muscularis mucosae and the invasion of the submucosa by isolated glandular elements of the mucosal epithelium. There was also moderate edema of the submucosa of the stomach. These gasinc changes were markedly accentuated in the two animals that were housed in metabolism cages and allowed to survive for two weeks. In these animals, the major changes in the stomach were associated with hypertrophy and hyperplasia of the gastric mucosa. There were large collections of gastric mucosal epithelium that had penetrated the tnuscuUi is mucosae forming large cysts and sheers of glandular epithelium throughout Die edematous sub mucosa. In addition, segments of the hyperplastic gastric mucosa were eroded thereby forming disrmci ulcers. / HONS 0857^ Tissue Modifications in Monkeys .tv Kci.ated to l>CB Table I. Hematological Changes in Monkeys hour Days Following PCB (Arodor 1248* j Administration 89 Assays conducted Hg (gm%> Hct (%) WBC (l03/cmn) Neutrophils (%) l ymphocytes (%) SCOT (Keitman-Frankel units/ml) SCPT (Reitman-F'rsnkel units/ml) BUN(mf%) TP (gm%) Control* I 3.8 i 0.9 43.1 + 3.2 6.3 i 1.8 44.5 t 12.0 54.4 t 12.0 43.0 4 13.3 24.9 i 6.9 19.8 5.4 7.4 + 0.6 PC B dosage* <3 |t/kg> ft. 5 g/ktf) 12.2 06' 39.0 1.99 9.9 2.4 63.0 * 16.0 37.0 * 15.2 1 9 ' U.6* 37.4 * 1 74 9.8 3 0 63.0 1 14.8* 36.0 i 14,6e 69.6 . 11.3 46.4 4 4.8 18.9 i 3.8 22.6 i 3.8 6.5 t 0.44 16.9 3.3' 19.4 t 2.1 6.5 t 0.34 Monsanto Company, St. Louis, Missouri. bVaiues expressed as mean I standard deviation, f Difference with controls statistically significant: p < .005 dDifference with controls statistically significant: p < 01 ^Difference with controls statistically significant , p < 05 The livers of the animals from the two experimental groups were slightly enlarged and comprised 2.8 percent of their body weight while the control livers were 2.1 percent. Ultrastni'ctural examination of the hepatic tissue demonstrated that the increase in size of the liver was due primarily to the proliferation of the smooth endoplasmic reticulum within the cytoplasm of the hepatic cells. The other cytoplasmic organelles and nuclei were unaltered. The liver homogenates of the experimental animals showed an increase in total RNA (Table II). However, due to the selected proliferation of the cytoplasmic endoplasmic reticulum, there was a decrease in the DNA content per gram of liver. Small modifications were also apparent in the enzyme activity of (he microsomal fraction (Table 111). There were significant decreases in glucose-6-phosphatase and esterase. The percent recovery of Aroclor 1248 that was added to control tissues and samples of facet and urine was: feeet, 98.7 t 14.3; urine. 83.3 i 6.3: kidney, 81.1 12.3. brain. 82.2 3.5; and liver, 94.81 4.2. The greatest percentage of the PCBs eliminated from the body occurred through biliary excretion into the gastrointestinal tract. By the 14th day, 5.60 percent of the original dose of the PCBs had been eliminated in the urine (0 38 mg) and feces (373 mg) I MQNS 085750 90 J. R. Alien ct al. from the animal that had received 3.0 g per kg body weight of PCB*, and 5 75 percent (0.88 mg in the urine and 237 mg in the feces) of the dose had been eliminated by the monkey that received 1.5 g per kg body weight. The chromatogram of the standard PCBs Table II. Biochemical Alterations In the Liver of Monkeys Given PC ft\ (Aroclor 1248) Control- PCB dosage-* (3 g/kg) H.Sg/kgj mg protein/mg DNA mg DNA/100 mg liver mg RNA/mg DNA 107 t 28 .346 .032 1.491.08 107 * 10 .2661 .014* 2.191.24* 98.8 * ! 1.0 .031* Values expressed as means 1 I standard deviation. bDifference with controls statistically significant: p < 01 `Difference with controls statistically significant: p < 0V Table HI. Hepatic Microsomal Ahcrutums in Monkeys Given PCBs (Aroclor 1248) " Control PCB dosage 3 B/ky 1 Si/kj Aniline hydroxylation (m^imol p-aminophenoi/30 min)b 11.1 3.9 10 0 2.0 H6i 40 Af-dem ethylation (mpmol formaldehyde/30 min)b 218 i 36 < 257 152 289 t 89 Nitroreduction (mffmol p-aminobenaoata/hr)b 16.0 3.3 21.5 . 8.5 24.3 9.8 Glucose-6-phosphatase (jimol PO4/15 min)b 1.71 t .37 1.13 1.18* 1.47 .18 Esterase Oimol p-mtrophanoi/min)b 3.44 .33 1.4 7 1 .I8d 3 37 .62 Values expressed as means a I standard deviation. bPer mg microsomal protein. 'Difference with controls statistically significant. p < .001 dDifference with controls statistically significant: p < .02. / M0NS 085751 Tissue Modifications in Monkeys as Related to PCB 91 (Aroclor 1248) containing numerous peaks having retention tunes between 40 and 325 sec. differed markedly from the chromatograms of the urinary residue obtained on the fourth day which contained an isolated peak at ! 20 see. and from that obtained from the urinary' residue of the 14th day which contained a major peak ai 120 sec (representing 93^ of the material) and minor peaks at 280 and 400 sec. Ihc chromatogram of the biliary residue contained five major peaks at 45. 80. 160. 300. ;;nd 355 sec (Figure I D) At (he time the animals were sacrificed on the fourth day levels of PCBs in the brain and kidney were about half those found in the liver (Tabic IV). However, in tissues taken from the two animals that survived fur (wo weeks there was a marked increase in liver PCBs and a decided decrease in levels of PCBs in the bruin and kidney. Tracings of chromatograms of the liver residues taken at four days and 14 days after PCD administra tion are depicted in Figure 1. B and C. At four days there is a greater percentage of isomers and perhaps polar metabolites having longer retention times than those found in the Fig. I. GLC-EC tracings of (A) Aroclor 1248; (B) the residues found in (he liver of a monkey 4 day*, and (C) 14 days after PCB administration; and (Dl the residue found in the gallbladder 14 days after PCB administration. / MONS 085752 92 J. R. Allen et j! Table IV. f.tvcls of PCB (Aroctor !24X\ in the Ttisuet <>f \l<tnkeyi* No. of animals 5 5 1 Days after exposure 4 4 14 Dose per kg body wt 1.5 .1.0 1.3 l.ivcr Jig/inn 24.0 i X .2 52.V * 27 7 V2.1 Kidney pg/gm 12.1 i 4.4* 27.2 *8.2 3.3 1 14 3.0 72.5 Values expressed as means t I standard deviation. ^Difference with liver value statistically significant p < .02. 1.8 Brain Mg/gm 16.7 i 7.3 28.1 t 7 5 Not detectable Not detectable standard (Figure 1. A). At 14 days, the chromatograms of the hepatic residue contain four major peaks with a greater proportion of the materia) having longer rcteniion limes than those found in the standard. Discussion These data indicate that a large percentage of the PCBs administered in this experiment is readily absorbed from the gastrointestinal tract and is subsequently slowly eliminated from the body. These findings are in agreement with the percentage absorption of PCBs by rodents recently reported by Albro and Pishbcm (1972). Probably as a result of the PCB metabolism and selective excretions, the chromatograms of the (issue residues and of the body excreta are modified from the chromatograms of the PCI) standard. The residues of the liver samples which contained greater prccentages of material with longer retention times suggest selective excretion of compounds having few chlorine atoms with the resultant hepatic residue consisting of a higher percentage uf highly chlorinated and more polar compounds. ^. Of the tissues examined for PCB content at four days after administration of the material, the liver contained the highest levels. In the two animals that were allowed to survive for 14 days, the hepatic levels of PCBs were higher than the hepatic levels at four days, while the kidney and brain levels decreased to very low levels. High levels of PCBs were reported by Grant et aL (1971) to be present in the rat adipose tissue and jt is asnimed that the fat depots of the monkeys also contained high concentrations of the PCBs. An explanation for the PCB location within the liver at 14 days is that PCBs from other tissues, including that from brain, kidney, and adipose tissue, have been mobilized and subsequently selectively taken up by the liver. During the period of reduced food intake following PCB administration, mobilization of lipid tissue containing the hydrophobic PCBs presumably occurred and resulted in the / MQNS 085753 Tissue Modifications in Monkeys as Related <o PCB 93 release of fatty acids and PCBs into the circulatory system. The continued exposure of the liver to the compounds and a suggested preferential hepatic uptake resulted in the higher hepatic levels observed at 14 days. The hematological changes that occurred m the animals Uuung the- four-day examina tion period were minimal. However, there were indications oi slight modifications in the hemoglobin and hematocrit values of the experimental animals. The moderate leuko cytosis and neutrophilia which occurred in this acute study are patterns observed in monkeys exposed to PCBs for longer periods. Monkeys fed lower levels of PCBs (or a period of three months were anemic and developed a decided leukocytosis (Allen ct ai. 1973). The increase in circulating white blood cells may be a response to the pustric irritation that develops in animals following exposure to the PCBs. Although not particu larly severe in the acute animals, ulcerations of the gastric mucosa and subsequent acute inflammatory responses may be sufficient to induce a leukocytosis. In addition, local hemorrhage in the gastric mucosa of the more chronically affected animals was apparent Hemorrhagic gastric ulceration would also account for a portion of the decrease in hemo globin and hematocrit that occurred in the PCB-intoxicated animals. There may also be an inhibiting effect of the PCBs on the hematopoietic tissue of the bone marrow and lymph tissue. The exact mechanism by which the PCBs produce these hypoplastic changes is not clear. Vos and Dc Roij (19721 have drown j decided decrease in antibody forming cells in the lymph nodes of guinea pigs tli.it had been exposed in the PCBs. In addition, there was a decrease in circulating lymphocytes in ihc PCB-fed animals (Vos 1972). The PCBs appear to have minimal immediate deleterious effects on liver function in the nonhuman primate, even when large doses are given, it has been shown in a number of animal species, including the monkey, that the PCBs are activators of microsomal drug metabolizing enzymes (Norback and Allen 1970: Alien and Abrahatnson 1972). Degen erative changes in the hepatic cells and focal liver necrosis are observed in monkeys that have been fed high levels of PCBs for extended periods (Allen el ui. 1973;. t The lesions that developed in the gastric mucosa arc possibly unique to (tie primate species. In the work that has been done on rodents (Allen and Abrahamson 1972). rabbits (Vos 1972). dogs (Keplinger et ai 1971), and other species (Vos and Koeman 1970), gastric hypertrophy, hyperplasia, or dysplasia of the mucosal epithelium have not been recorded. However, in human outbreaks of PCB intoxication, nausea and vomiting, both of which are consistent with gastric irritation, were a constant observation (Kuratsune 1969). The presently reported research indicates that a single large dose of the PCBs to non human primates is capable of producing injurious effects particularly to the gastric mucosa after short periods of time. Because of the high degree of absorption and sub sequent tissue deposition, as well as the slow elimination from the body, large amounts of the PCBs remain in the body for long periods. Evidence suggests they are gradually / MQNS 085754 94 J. R. Alien cl al mobilized from (he tissue stores, sequestered in the liver, and eliminated by biliary ex cretion. Mobilization of the PCUs ensures a continuing exposure of tissues to low level PCBs for an extended |>eriod. Acknowledgments The authors thank L. J. Abrahamson. L. A. Carstens, and J. M. Scliefller for their technical assistance. References AJbro, P. W., and L. Fishbein: Intestinal absorption of polychlorinated biphenyls in rats. Bull. Environ. Contamin. Toxicol. B, 26 (1972). Allen, J. R., and L. J. Abrahamson: Enzymatic changes in the liver of rats fed poly chlorinated biphenyls, triphenyls, and DDT. Croc. Amer. Chcm. Soc., Div. Water, Air, and Waste Chem. 12,97(1972). Allen, J. R., L. J. Abrahamson, and D. H. Norback: Biological eflecis of chlorinated biphenyls and triphenyls on the subhuman primaic. Environ. Res. 6, 344 (1973). Armed Forces Institute of Pathology, Manual of Histologic and Special Staining Techniques. New York, McGraw-Hill (I960). Beckman Model R-101 Microzone Electrophoresis Cell instruction Manual RM-1M-3, (1965). Bohoun, C., J. C. Delarue, and E. Comoy: Direct method for the determination of urea in blood with diacetyl monoxime. Clin. Chirn. Acta 18,417 (1968). Caulfield, J. B.: Effects of varying the vehicle for 0s04 in tissue fixation. .1. Uiophys. Biochem. Cytol. 3,827(1957). ' Curley. A., V. W. Burse, M. E. Grim, R. W. Jennings, and R. I7.. Linder: Polychlorinated biphenyls: Distribution and storage in body fluids and tissues of Sherman rats. Environ. Res. 4,481 (1971). Grant, D. L.. W. E. J. Phillips, and D. C. Villcneuve: Metabolism of a polychlorinated biphenyl (Aroclor 1254) mixture in the rat. Bull. Environ. Contamin. Toxicol. 6, 102(1971). Holmes, D. C., J. H. Simmons, and J. O'G. Tatton: Chlorinated hydrocarbons in British wildlife. Nature 216. 227 (1967). Jennings, R. W.: Biological sample clean-up for chlorinated hydrocarbon and organophosphorus analysis using electron-capture gas-liquid chromatography. Paper pre sented at Am. Chem. Soc. Meeting, Atlantic City. New Jersey (1968). Jensen, S., A. G. Johnels, M. CHsson, and G. Otterlind: DDT and PCB in marine animals from Swedish waters. Nature 224, 247 (1969) Keplinger, M. L., O. E. Fancher, and J. C. Calandra: Toxicological studies with chlorinated / MONS 085755 Tissue Modifications in Monkeys us Related to PCB 9$ . biphenyls. Proceedings NIEHS Polychlorinated Biphenyl Conference, Research Triangle Park, N. C., Dec. 20-21 (1971). Koeman, J. H., A. A. C. Oskamp, J. Veen, E. Brouwer, J. Rooth, P. Zwart, E. V D Broek, and H. Van Genderen: Insecticides as a factor in the mortality of the sandwich tern (Sterna sandviccnsis). Meded. Rijksfac. Landbouwwctcnschappcn Gent. 32, 841 (1967). Koeman, J. H., M. C. Ten Noever De Brauw, and R. H. l)e Vos: Chlorinated biphenyls in fish, mussels, and birds from the River Rhine and the Netherlands coastal area. Nature 221, 1126(1969). ,Kuratsune, M.: An epidemiologic study on "Yusho" or chlorobiphenyk poisoning. Fukuoka Acta Medica 60 513 (1969). Kuratsune, M., T. Yoshimura, J. Matsu/aka, and A. Yamaguchi: Epidemiologic study on Yusho, a poisoning caused by ingestion of rice oil contaminated with a commercial brand of polychlorinated biphenyls. Environ. Health 1`crsp. t, 119 (1972;. Lowry, 0. H., N. J. Rotebrough, A. L. Farr, and R. J. Randall: Protein measurement with Folin phenol reagent. J. Biol.Chem. 193, 265(1951). Mollenhauer, H. H.: Plastic embedding mixture for use in electron microscopy. Stain Tech. 39. Ill (1964). Munro, H. M., and A. J. Fleck: The determination of nucleic acids. Methods of Uiochem. Anal. 14,113(1966). Norback, D. H., and J. R. Allen: Enzymatic and morphologic alterations of hepatic endoplasmic reticulum induced by chlorinated aromatic hydrocarbons, fed. Proc. 29,816(1970). Norback, D. H., and J. R. Allen: Chlorinated triphenyi induced extension of the hepatic endoplasmic reticulum.Proc.Soc. Exp. Biol. Med. 139, 1127(1972). Reitman, S., and S. Frankel: A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases Am. J. Clin. Path 28, 56 (1957). Vos, J. G.: Toxicology of the PCBs for mammals and turds, Environ. Health Persp. I, 105(1972). *' Vos, J. G., and J. H. Koeman: Comparative toxicologic study with polychlorinated bi phenyl in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues. Toxicol. Appl. Pharmacol. 17, 656 (1970) Vos, J. G,, and T. De Roij: Immunosuppressive activity of a polychlorinated biphenyl ,preparation on the humoral immune response in guinea pigs. Toxicol Appl. Pharmacol. 21 549(1972). Werchselbaum, T. E.: An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Am. J. Clin. Path. 28, 56 (1946). Manuscript received July 12, 1973: accepted October 28, 1973 / HUNS 085756